EXTENSIVE POLYMORPHISM AT THE GP63 LOCUS IN-FIELD ISOLATES OF LEISHMANIA-PERUVIANA

Genetic,diversity within and between tandemly arrayed copies of the Gp 63 gene occurs in laboratory isolates of Leishmania spp., but the exte nt to which this represents natural genetic diversity has not been ass essed. Here, the Gp63 focus is examined in 58 fresh isolates of L. per uviana, and clones derived from them, collected throughout the Peruvia n Andes. Extensive polymorphism is observed, both in size of Gp63 cont aining chromosomes, and for restriction-fragment-length polymorphisms (RFLPs) at the Gp63 locus. All clones within an isolate are identical, including those with two distinct Gp63-hybridising chromosomal-sized pulsed-field gel electrophoresis (PFGE) bands, consistent with diploid y but with size differences in homologous chromosomes. For RFLP analys is, three enzymes were selected to cut within the coding region (PstI) , in the intergenic region (SalI) and outside (EcoRI) the Gp63 gene cl uster. PstI gave identical banding patterns across all isolates/clones . For EcoRI and SalI, all clones within an isolate were identical, but isolates were polymorphic for fragments at 13 (2-30 kb) and 8 (2.6-8. 8 kb) different molecular mass locations generating 19 and 16 distinct RFLP patterns or genotypes for each enzyme, respectively. EcoRI restr iction patterns, analysed by PFGE, were consistent with the presence o f two clusters of Gp63 genes on each homologous chromosome, one contai ned within EcoRI fragments large enough to carry from 3 to 10 copies o f the Gp63 gene, the second on fragments which could carry 1 or 2 copi es of the gene. SalI patterns indicated variable restriction sites wit hin clusters, but not within every intergenic region. A hierarchical a nalysis of variance of allele frequencies, expressed in terms of Wrigh t's F-statistic, indicated significant barriers to gene flow at all le vels, valleys within regions (north/south), villages within valleys, a nd individuals within villages. This extreme polymorphism at the Gp63 locus of L. peruviana demonstrates the great potential for generation of genetic diversity in parasite populations.